Telomeres and telomerase play a critical role in cellular aging and human carcinogenesis. Telomere shortening, telomerase activation, and genetic instability are among the best-characterized changes observed in age-associated senescence and pre-malignant neoplastic cells. In normal cells, telomere attrition is one mechanism that controls proliferative capacity and replicative aging. In most cancer cells, elevated telomerase levels bypass these normal constraints on telomere-associated growth regulation. Telomerase activity is regulated not only by the expression of its protein-components (via growth stimulation and oncogene signaling), but also by its access to telomere DNA and dynamic changes in the nucleoprotein structures formed at telomeres. The telomere-repeat binding factors (TRFs) function to prevent DNA recombination and DNA damage responses at normal telomeres. One of the functions of TRFs and their associated factors (termed shelterins) is to establish a chromatin structure that limits DNA recombination and other forms of DNA damage, yet is sufficiently dynamic to allow telomerase access to critically short telomeres each cell cycle. Recently, a non-coding RNA containing telomere repeats (referred to as TERRA) has been identified as a structural component of the shelterin complex. We have found that TERRA binds to key components of the shelterin complex and that it promotes heterochromatin formation through interactions with heterochromatin protein 1 (HP1), histone K3 K9me3, and the Origin Recognition Complex (ORC). TERRA abundance correlates inversely with telomerase expression and TERRA RNA can inhibit telomerase activity in vitro. TERRA expression is also suppressed in most telomerase positive cancer cells. Our preliminary data indicates that TERRA expression is induced by inhibition of cellular proliferation. The precise function of TERRA and its potential role as a telomere and telomerase regulatory molecule are just beginning to be explored in detail. In this proposal, we plan to test the hypothesis that TERRA is a central regulatory molecule that coordinates telomere heterochromatin formation with telomere length and cellular proliferation. The long-term goal of this proposal is to determine how telomeric heterochromatin is deregulated in cellular aging and cancer. PUBLIC HEALTH RELEVANCE: Telomere dysfunction is an early biomarker for dysplastic and pre-cancerous lesions. Telomere shortening and loss of telomere signals are commonly observed in early stage carcinogenesis, but their cause and contribution to cancer cell evolution is not known. In this application, we investigate the novel role of telomere heterochromatin in maintaining telomere stability and how this may be deregulated in human cancer. We will examine telomere chromatin alterations during cellular ageing and after immortalization of primary B-lymphocytes. These studies will provide new information on our understanding of telomere chromatin structure and genetic stability during the early stages of cancer cell transformation and normal cell senescence.